Assays for B Lymphocyte Function

Subbarao Bondada1, Darrell A. Robertson1

1 University of Kentucky, Lexington, Kentucky
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.8
DOI:  10.1002/0471142735.im0308s56
Online Posting Date:  November, 2003
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This unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. The first basic protocol is a generalized system for inducing in vitro antibody production and can accommodate various types of antigens under study. Secreted antibodies can then be measured with an enzyme‐linked immunosorbent assay (ELISA) or other soluble‐antibody detection systems. Alternatively, the number of antibody‐producing cells can be quantified by plaque‐forming cell (PFC) assays presented in this unit: the Cunningham‐Szenberg and the Jerne‐Nordin techniques. Both methods employ specially prepared slide chambers, described here, in which the antibody‐producing B cells are mixed with complement and indicator sheep red blood cells (SRBC), or with trinitrophenol‐modified SRBC (TNP‐SRBC), with subsequent lysis and counting of plaques. Because IgM antibodies fix complement efficiently, whereas IgG and IgA antibodies do not, unmodified PFC assays measure only IgM antibodies. The assay can be modified, however, to measure all classes of antibodies or to enumerate total immunoglobulin‐secreting B cells, as described in alternate protocols. Yet another method of measuring the number of antibody‐producing B cells (in a class‐specific fashion) is to use the ELISPOT technique described in UNIT 7.14. The resting B cells used in these procedures are prepared as described in the final support protocols for Percoll gradient centrifugation.

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Table of Contents

  • Assays for B Cell Function
  • Basic Protocol 1: Antigen‐Specific Antibody Production
  • Plaque‐Forming Cell Assays
  • Basic Protocol 2: Jerne‐Nordin PFC Assay
  • Basic Protocol 3: Cunningham‐Szenberg PFC Assay
  • Alternate Protocol 1: Modifying the Jerne‐Nordin PFC Assay for Isotype‐Specific Responses
  • Alternate Protocol 2: Modifying the Jerne‐Nordin PFC Assay to Measure Polyclonal Antibody Responses
  • Support Protocol 1: Preparation of Agarose‐Coated Slides
  • Support Protocol 2: Preparation of Cunningham‐Szenberg Chambers
  • Support Protocol 3: Coupling Trinitrophenyl (TNP) Hapten to SRBCs
  • Support Protocol 4: Protein A–Coupled SRBC
  • Support Protocol 5: Preparation of TNP‐Ovalbumin
  • Support Protocol 6: Purification of Resting B Cells by Percoll Gradient Centrifugation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Antigen‐Specific Antibody Production

  • 2× RPMI 1640 with bicarbonate (Invitrogen)
  • SeaPlaque low‐melting‐point agarose (FMC BioProducts)
  • TNP‐SRBC (see protocol 8)
  • Guinea pig complement (Colorado Serum)
  • DPBS (see reciperecipe)
  • 0.85% (w/v) NaCl (i.e., saline)
  • 43°C water bath
  • 12 × 75–mm borosilicate glass tubes (Fisher)
  • Agarose‐coated slides (see protocol 6)
  • Gauze
  • Slide trays, custom made (see recipe and Fig. )
  • Cloth
  • Slide rack and glass staining jar
  • Low‐power (10×) dissecting binocular microscope with indirect light source
NOTE: Before the PFC assay can be started, the glass slides have to be precoated with 0.15% agarose and the SRBC have to be coupled with the desired antigen when hapten‐coupled TI or TD antigens are used (see Support Protocols).

Basic Protocol 2: Jerne‐Nordin PFC Assay

  • Wax, tissue‐preparation grade
  • Supplemented complete RPMI or IF12 medium (see reciperecipes)
  • 7.5% SRBC or 15% TNP‐SRBC (see protocol 8)
  • Washed B cells from cultures (see protocol 1)
  • 50% guinea pig complement (e.g., Life Technologies, Colorado Serum) in complete medium
  • 120‐cm2 glass petri dish
  • 96‐well U‐bottom microtiter plates
  • Cunningham‐Szenberg chamber (see protocol 7)
  • Slide tray (e.g., from a CO 2 incubator)
  • Dissecting microscope with 10× magnification (optional)
NOTE: All reagents should be warmed to room temperature to avoid air bubble formation inside the chambers during incubation.

Basic Protocol 3: Cunningham‐Szenberg PFC Assay

  • 70% ethanol
  • SeaPlaque low‐melting‐point agarose (FMC Bioproducts)
  • Glass microscope slides frosted on one end (e.g., Gold Seal Rite‐ON micro slides, Fisher)
  • Slide racks and staining jars (Fisher)
  • Hot air oven
  • 100‐ml glass bottle
  • 43°C water bath
  • Slide tray (see recipe)
  • Paint brush (optional)
  • Slide box

Alternate Protocol 1: Modifying the Jerne‐Nordin PFC Assay for Isotype‐Specific Responses

  • 70% ethanol
  • 3‐in. × 1‐in. × 0.93‐ to 1.05‐mm glass microscope slides
  • Slide racks and staining jars (Fisher)
  • Hot air oven
  • 0.25‐in. double‐sided tape (e.g., 3M)
  • Roller (e.g., 25‐ml pipet or similar round object)

Alternate Protocol 2: Modifying the Jerne‐Nordin PFC Assay to Measure Polyclonal Antibody Responses

  • Glycylglycine
  • 1× MBB (see recipe)
  • 2,4,6‐trinitrobenzenesulfonic acid (picryl sulfonic acid, TNBS), sodium salt
  • 0.28 M cacodylate buffer, pH 6.9 (see recipe)
  • Sheep's blood obtained from a single animal, <4 weeks old
  • 15‐ml graduated conical tube
  • 10‐ml syringe and 18‐G needle

Support Protocol 1: Preparation of Agarose‐Coated Slides

  • Sheep red blood cell (SRBC) suspension (Colorado Serum)
  • Saline: 0.15 M NaCl (Life Technologies)
  • 1.0 mg/ml Protein A in saline (Pharmacia or Sigma‐Aldrich)
  • Chromium (III) chloride hexahydrate
  • 15‐ml conical centrifuge tube
  • Rocker Platform
NOTE: Phosphate‐buffered saline (PBS) should not be used in any step of this protocol. This is to avoid formation of chromium phosphate precipitate.

Support Protocol 2: Preparation of Cunningham‐Szenberg Chambers

  • Ovalbumin (Sigma‐Aldrich)
  • 0.1 M sodium bicarbonate solution (see recipe)
  • TNBS
  • 1× saline
  • Aluminum foil
NOTE: The calculations in this unit assume that 1.0 mg/ml ovalbumin has an OD 280 of 0.587 and that TNBS has a molar extinction coefficient of 1.25 × 104 M1 cm 1

Support Protocol 3: Coupling Trinitrophenyl (TNP) Hapten to SRBCs

  • 1× and 10× Hanks balanced salt solution (HBSS), calcium and magnesium free (Life Technologies), 4°C
  • Percoll (Pharmacia LKB or Sigma‐Aldrich)
  • 1 M HEPES
  • 1 N HCl
  • 15‐ and 50‐ml polypropylene centrifuge tubes
  • Sorvall RT6 tabletop centrifuge and H‐1000B rotor or equivalent
  • Additional reagents and equipment for preparing cells (units 3.1), depleting T cells (units 3.4), and counting cells ( 3.NaNappendix 3)
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Literature Cited

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